Page 273 - Plant Canada 2024 Proceeding

PLANT CANADA 2024

be compared to databases to identify the bacteria and fungi to different levels of taxonomic resolution,
depending on which variable regions of the rRNA are sequenced. Most studies that perform rRNA
analysis use short-read sequencing technologies that can target a limited number of variable regions due
to restrictions on sequencing read length. Recently, the long-read sequencing platform offered by PacBio
has enabled the sequencing of several variable regions on a single reaction, increasing the resolution of
taxonomic identification. New advances in PacBio sequencing have also increased throughput, with the
release of the high-yield PacBio Kinnex full-length RNA kit. In this study, we applied the Kinnex rRNA kit
to long (1200-1500 bp) and short length (300-450 bp) 16S and ITS rRNA regions, respectively, to the
same soil microbiome DNA samples. In order to perform ITS sequencing as well as shorter amplicon
sequencing, we modified the Kinnex protocols and adapted them for sequencing different amplicons. The
DNA samples were extracted from soil obtained from different field crops grown in Saskatoon,
Saskatchewan. We observed differences in DNA quality (intactness and yield) and microbiome profiles
based on different crop types, DNA extraction methods, and amplicon length. Using these latest state-of-
the-art approaches, we are able to identify both bacteria and fungi within complex soil communities with
high resolution and throughput.

*[P140] LOCALIZATION OF ALKALOID BIOSYNTHETIC ENZYMES IN LOPHOPHORA WILLIAMSII
CACTUS. Ginny Li and Peter J. Facchini. University of Calgary, 2500 University Drive N.W., Calgary, AB,
Canada, T2N 1N4
Correspondence to: ginny.li@ucalgary.ca, pfacchin@ucalgary.ca

Peyote (Lophophora williamsii) is a cactus known to the phenethylamine protoalkaloid mescaline.
Consuming peyote ‘buttons’ has been used in Indigenous ceremonies for at least six millennia owing to
the psychedelic effect of mescaline. Recently, hallucinogenic compounds such as psilocybin have been
used to treat mental health disorders including depression, anxiety, or post-traumatic stress disorder.
Mescaline is currently undergoing clinical assessment for its potential therapeutic benefits.

The biosynthetic pathway converting L-tyrosine to mescaline, and related phenethylamines and
tetrahydroisoquinoline (THIQ) alkaloids, was recently elucidated in our laboratory. L-Tyrosine is first
hydroxylated by a cytochrome P450 (LwCYP76AD94) to form L-DOPA, which is then decarboxylated by a
tyrosine/DOPA decarboxylase (LwTYDC) to yield dopamine. A specific S-adenosylmethionine-dependent
O-methyltransferase (LwOMT2) subsequently produces 3-methoxytyramine, which undergoes
hydroxlation and subsequent O-methylation at the 5 position and a final O-methylation by LwOMT10 at
the 4 position to yield mescaline. The diversity of metabolites associated with this core pathway include
THIQ alkaloids resulting from the Pictet-Spengler condensation of phenethylamines, and N-methylated
derivatives of corresponding phenethylamine and THIQ compounds formed via a unique N-
methyltransferase (LwNMT).

Polyclonal antibodies were raised against LwTYDC, LwOMT10 and LwNMT as tools to investigate the
cellular localization of phenethylamine and THIQ biosynthesis in peyote. Based on based on the use of
matrix-assisted laser desorption/ionization mass spectrometry imaging, mescaline was reported to
accumulate primarily in, or near, the epidermis of aerial organs. Immunoblot analyses of total protein
extracts from shoots sectioned longitudinally or cross-sectionally detected all three enzymes in all parts of
the aerial organs, with the relative abundance increasing from bottom to top, and from the inside out.
Liquid chromatography-mass spectrometry (LC-MS) analysis of corresponding sections showed that the
phenethylamine and THIQ content was generally higher in apical and peripheral tissues, although
mescaline levels were relatively consistent in regions of the shoot. Total RNA was also extracted from
aliquots of each sectional sample to perform qRT-PCR, which showed that, in contrast to the
immunolocalization results, LwOMT10 and LwNMT transcript levels were highest in the apex, but
declined sharply in other areas of the shoot, whereas LwTYDC transcripts showed an inverse localization
pattern with higher levels in basal region.

Immunolocalization in fixed tissue sections showed that LwTYDC and LwNMT are associated with
chloroplasts, whereas LwOMT10 is associated with the nucleus. Sucrose density gradient fractionation
followed by immunoblot analysis of each fraction confirmed that LwOMT10 and LwNMT were associated
with cellular fractions with the same density as nuclei and chloroplast, respectively, although both

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